Real-Time, Ultrasensitive, and High-Throughput Monitoring of Adeno-Associated Virus Production via an Aldehyde-Functionalized Dual-Capture MetaSPR Fishing Sensor

Adeno-associated virus (AAV) is a key vector in gene therapy, yet conventional AAV quality control methods are time-consuming and unsuitable for real-time monitoring, severely hindering process optimization. Here, we present an aldehyde-functionalized metasurface plasmon resonance (ALMetaSPR) biosensor for rapid, label-free, and high-throughput AAV quantification. Two complementary platforms were developed: a 96-well high-throughput system enabling analysis within 15 min (8-fold faster than ELISA); and a portable MetaSPR-based process analytical technology (PAT) device (MSPR-PAT) that integrates microfluidics and smartphone interfaces for real-time online monitoring of AAV production. Both platforms can accurately quantify AAV-2 and AAV-5 titers directly without sample labeling. The antibody-based capture system exhibited a broad dynamic range (6.25 × 10⁰⁹-4.00 × 10¹¹ GC/mL) with a limit of detection (LOD) of 1.24 × 10⁰⁹ GC/mL, representing a 250-fold sensitivity improvement over current label-free techniques. The biosensor surface can be regenerated more than 42 cycles without performance loss, which significantly reduces the per-test costs. This innovation resolves a critical bottleneck in gene-therapy manufacturing by integrating high-throughput screening, real-time process analytics, and one-step operation.